Bottom Line:
In addition, we have tested the effects of inhibitors on mouse Nat2, including compounds which are endogenous and exogenous steroids.We show that tamoxifen, genistein and diethylstilbestrol inhibit mouse Nat2.We propose that a conformational change in the structure is required in order for ligands to bind to the active site of the protein.

ABSTRACTThere is increasing evidence that human arylamine N-acetyltransferase type 1 (NAT1, EC 2.3.1.5), although first identified as a homologue of a drug-metabolising enzyme, appears to be a marker in human oestrogen receptor positive breast cancer. Mouse Nat2 is the mouse equivalent of human NAT1. The development of mouse models of breast cancer is important, and it is essential to explore the biological role of mouse Nat2. We have therefore produced mouse Nat2 as a recombinant protein and have investigated its substrate specificity profile in comparison with human NAT1. In addition, we have tested the effects of inhibitors on mouse Nat2, including compounds which are endogenous and exogenous steroids. We show that tamoxifen, genistein and diethylstilbestrol inhibit mouse Nat2. The steroid analogue, bisphenol A, also inhibits mouse Nat2 enzymic activity and is shown by NMR spectroscopy, through shifts in proton peaks, to bind close to the active site. A three-dimensional structure for human NAT1 has recently been released, and we have used this crystal structure to generate a model of the mouse Nat2 structure. We propose that a conformational change in the structure is required in order for ligands to bind to the active site of the protein.

fig3: Comparison of the crystal structures of NAT from M. smegmatis (MSNAT) and human NAT1. The surface shown was calculated from the MSNAT crystal structure (PDB accession code 1GX3), and the human NAT1 crystal structure F125S mutant (PDB accession code 2IJA) is shown in ribbon format. The C-terminus and inter-domain loop region of human NAT1 protrude from the eukaryotic protein core, and both of these protein regions block the prokaryotic NAT active site. The C-terminus of human NAT1 is found in the region termed the ‘β-site’ [47].

Mentions:
Until recently, there has been no structural information on eukaryotic NAT enzymes. However, recently the three-dimensional structure of a human NAT1 mutant (Phe/Ser substitution at position 125) was determined by X-ray crystallography (PDB accession code: 2IJA [30]). A non-mutated human NAT1 crystal structure has also been deposited in which the active-site cysteine residue was modified to S-(2-anilino-2-oxoethyl)-cysteine (PDB accession code: 2PQT). It is considered likely that both the mutation of residue 125 and the modification of the active-site cysteine residue stabilise the structure of the human enzyme, because thousands of attempts to generate a crystal from the native human NAT1 protein have been unsuccessful (A. Kawamura, unpublished results). The structure of human NAT1 is illustrated in comparison with the NAT from M. smegmatis (Fig. 3). The eukaryotic proteins contain a loop between the second and third domains. This inter-domain loop is not present in their prokaryotic counterparts [31], as highlighted in Fig. 3. It is interesting to note that in the structure of the human NAT1 enzyme, this inter-domain loop is folded back over the active site. Likewise, the C-terminus is shown to be folded over the active-site cleft. These observations indicate that a conformational change in the protein may be required to allow the substrates to access the active-site cysteine residue. However, a structure of human NAT2 with coenzyme A bound has also been deposited (PDB accession code: 2PFR), and the position of the coenzyme A is such that the C-terminus and inter-domain loops regions are not significantly changed. This is in contrast to the recently reported structure of Mycobacterium marinum NAT in complex with coenzyme A [32]. In this bacterial NAT, the C-terminus is shorter than in the eukaryotic structures, and there is no inter-domain loop, and the ligand is found in the space where these regions are located in the eukaryotic structure.

fig3: Comparison of the crystal structures of NAT from M. smegmatis (MSNAT) and human NAT1. The surface shown was calculated from the MSNAT crystal structure (PDB accession code 1GX3), and the human NAT1 crystal structure F125S mutant (PDB accession code 2IJA) is shown in ribbon format. The C-terminus and inter-domain loop region of human NAT1 protrude from the eukaryotic protein core, and both of these protein regions block the prokaryotic NAT active site. The C-terminus of human NAT1 is found in the region termed the ‘β-site’ [47].

Mentions:
Until recently, there has been no structural information on eukaryotic NAT enzymes. However, recently the three-dimensional structure of a human NAT1 mutant (Phe/Ser substitution at position 125) was determined by X-ray crystallography (PDB accession code: 2IJA [30]). A non-mutated human NAT1 crystal structure has also been deposited in which the active-site cysteine residue was modified to S-(2-anilino-2-oxoethyl)-cysteine (PDB accession code: 2PQT). It is considered likely that both the mutation of residue 125 and the modification of the active-site cysteine residue stabilise the structure of the human enzyme, because thousands of attempts to generate a crystal from the native human NAT1 protein have been unsuccessful (A. Kawamura, unpublished results). The structure of human NAT1 is illustrated in comparison with the NAT from M. smegmatis (Fig. 3). The eukaryotic proteins contain a loop between the second and third domains. This inter-domain loop is not present in their prokaryotic counterparts [31], as highlighted in Fig. 3. It is interesting to note that in the structure of the human NAT1 enzyme, this inter-domain loop is folded back over the active site. Likewise, the C-terminus is shown to be folded over the active-site cleft. These observations indicate that a conformational change in the protein may be required to allow the substrates to access the active-site cysteine residue. However, a structure of human NAT2 with coenzyme A bound has also been deposited (PDB accession code: 2PFR), and the position of the coenzyme A is such that the C-terminus and inter-domain loops regions are not significantly changed. This is in contrast to the recently reported structure of Mycobacterium marinum NAT in complex with coenzyme A [32]. In this bacterial NAT, the C-terminus is shorter than in the eukaryotic structures, and there is no inter-domain loop, and the ligand is found in the space where these regions are located in the eukaryotic structure.

Bottom Line:
In addition, we have tested the effects of inhibitors on mouse Nat2, including compounds which are endogenous and exogenous steroids.We show that tamoxifen, genistein and diethylstilbestrol inhibit mouse Nat2.We propose that a conformational change in the structure is required in order for ligands to bind to the active site of the protein.

ABSTRACTThere is increasing evidence that human arylamine N-acetyltransferase type 1 (NAT1, EC 2.3.1.5), although first identified as a homologue of a drug-metabolising enzyme, appears to be a marker in human oestrogen receptor positive breast cancer. Mouse Nat2 is the mouse equivalent of human NAT1. The development of mouse models of breast cancer is important, and it is essential to explore the biological role of mouse Nat2. We have therefore produced mouse Nat2 as a recombinant protein and have investigated its substrate specificity profile in comparison with human NAT1. In addition, we have tested the effects of inhibitors on mouse Nat2, including compounds which are endogenous and exogenous steroids. We show that tamoxifen, genistein and diethylstilbestrol inhibit mouse Nat2. The steroid analogue, bisphenol A, also inhibits mouse Nat2 enzymic activity and is shown by NMR spectroscopy, through shifts in proton peaks, to bind close to the active site. A three-dimensional structure for human NAT1 has recently been released, and we have used this crystal structure to generate a model of the mouse Nat2 structure. We propose that a conformational change in the structure is required in order for ligands to bind to the active site of the protein.